15dPGJ2 reduces NF-κB in peripheral blood mononuclear cells and reduces the production of the pro...

15dPGJ2 reduces NF-κB in peripheral blood mononuclear cells and reduces the production of the pro inflammatory

cytokines IFN-γ and TNF-α in T helper cells

Lynne Sykes1, David MacIntyre1, Sathana Ponnampalam1, Xiao J Yap1, TG Teoh2, and Phillip R Bennett1.

1IRDB, Imperial College, London, United Kingdom, 2Department of Obstetrics and Gynaecology, Imperial College

Healthcare NHS Trust, London, United Kingdom

Preterm labour

• Preterm birth occurs in 8% -12% of pregnancies

• Infection/Inflammation strongly associated with early preterm labour occurring in 80% of SPTB < 28 weeks 1

• Inflammation leads to the activation of pro-labour gene expression 2

• Association between inflammation and neonatal brain injury 3

• Despite increased awareness of this association (Figure 1) this has not translated into a reduction in PTB or neonatal brain injury4

1 Goldenberg RL, Andrews WW, Hauth JC. Choriodecidual infection and preterm birth. Nutr Rev 2002;60(5 Pt 2):S19-252 Lindstrom TM, Bennett PR. The role of nuclear factor kappa B in human labour. Reproduction , 2005;130(5):569-81.3 Gotsch et al. The fetal inflammatory response syndrome Clin Obstet Gynecol, 2007; 50(3):652-83.4 Sykes et al. Targeting Immune Activation in the Prevention of Pre-term Labor. US Obstetrics & Gynecology,2011;6(2):103-9.

Figure 1Number of publications with reference to preterm labour and inflammation 4

(Sykes et al 2012, Targeting Immune activation for the prevention of Preterm labour)

Triggers of NF-κB activation include pro-inflammatory cytokines e.g. TNF- α, IL-1β 1

Inflammation and labour

Figure 2 NF-κB controls the expression of labour associated genes MMP’s COX-2, IL-8.

NF-κB is activated by pro inflammatory proteins which can thus trigger infection induced preterm labour.

Lindstrom TM, Bennett PR. The role of nuclear factor kappa B in human labour. Reproduction , 2005;130(5):569-81.

NF-κB is a pro inflammatory transcription factor which also controls the expression of labour associated genes (MMP’s, COX-2, IL-8)1

NF-κB DNA binding activity is increased in the myometrium and amnion at term and in labour respectively 5, 6 (Chapman et al 2004 J Clin Endocrinol Metab, Allport et al 2001 Molecular Human Reprod)

Inflammation and neonatal morbidity

Fetal inflammatory response syndrome

Originally described in PTL/PPROMDefined by an elevated IL-6 level in fetal plasmaSystemic activation of the fetal innate immune system 7

Target organs and effects: Adrenal gland- increase in cortisol Lungs – increase in chronic lung diseaseBrain – increase in PVL, IVH

Figure 3. Intrauterine infectionPictorial representation of the ascending and transplacental routes of intrauterine infection. Romero R, Mazor M. Infection and preterm labor. Clin Obstet Gynaecol 1982;9:593–607

Figure 4. Target organs of the Fetal inflammatory response syndrome 7Gotsch et al. The fetal inflammatory response syndrome Clin Obstet Gynecol, 2007; 50(3):652-83.

Inflammation and neonatal morbidity

Fetal inflammatory response syndrome

Originally described in PTL/PPROMDefined by an elevated IL-6 level in fetal plasmaSystemic activation of the fetal innate immune system 7

Target organs and effects: Adrenal gland- increase in cortisol Lungs – increase in chronic lung diseaseBrain – increase in PVL, IVH

Figure 3. Intrauterine infectionPictorial representation of the ascending and transplacental routes of intrauterine infection. Romero R, Mazor M. Infection and preterm labor. Clin Obstet Gynaecol 1982;9:593–607

Figure 4. Target organs of the Fetal inflammatory response syndrome 7Gotsch et al. The fetal inflammatory response syndrome Clin Obstet Gynecol, 2007; 50(3):652-83.

Immunology of pregnancy

Figure 5. Regulation of Th1:Th2 interleukin production A negative interaction exists between the Th1 and Th2 interleukins. IFN-γ inhibits Th2,whereas IL-4 inhibits Th1 interleukins.

Adapted from O’Garra et al. The molecular basis of T helper 1 and T helper 2 cell differentiation. Trends in Cell Biology, 2000

“Immune paradox”: Balance between immune tolerance of the foreign fetus along with maintaining defence against infection

Altered bias of Th1:Th2 ratio to favour Th2 profile thought to contribute to the maintenance of successful pregnancy

Progesterone and Prostaglandin D2 promote the Th2 profile

NF-κB is suppressed in T cells in pregnancy, which is thought to contribute to suppression of Th1 interleukins 8

(McCracken et al 2004 Journal of Immunology)

The cyclopentenone prostaglandin: 15dPGJ2Anti-inflammatory prostaglandin

• Inhibits NF-κB and COX-2 in amniocytes and myocytes in vitro

• Independent of PPAR-γ

• Delays preterm labour in the murine model of inflammation induced preterm labour

• Improves pup mortality

Figure 6. Western blots of NF-κB and COX-2 15dPGJ2 reduces NF-κB and COX-2 in IL-1β stimulated myocytes in culture. Similar results were seen with amniocytes. Lindstrom et al 2005. Journal of Clin Endocrinol Metab 90: 3534-3543.

Figure 7. Murine model of infection induced preterm labour15dPGJ2 delays LPS induced preterm delivery and increases pup survivalPirianov et al 2009. Endocrinology 150:699-706

The cyclopentenone prostaglandin: 15dPGJ2CRTH2 agonist

• CRTH2 is the second Prostaglandin D2 receptor

• Classic receptor for Th2 cells

• PGD2 stimulates Th2 interleukin production in vitro in Th2 cells 9

• Potential for promotion of the Th2 response in pregnancy and suppression of the pro inflammatory Th1 response .

Figure 8. Effect of Prostaglandin D2 on Th2 interleukinsA dose response increase in IL-4 and IL-10 production is seen with Prostaglandin D2 in cultured Th2 cells. 9 Xue et al, Prostaglandin D2 causes preferential induction of pro inflammatory Th2 cytokine production through an action on CRTH2 on Th2 cells. Journal of Immunology 2005; 175; 6531-6536

• Potential for promotion of the Th2 response in pregnancy and suppression of the pro inflammatory Th1 response .

• Potential for promotion of the Th2 response in pregnancy and suppression of the pro inflammatory Th1 response.

Hypothesis and aims

• Hypothesis15dPGJ2 represents a potential anti-inflammatory therapeutic agent for the prevention of infection induced preterm labour and inflammation induced neonatal brain injury.It is not known what effects 15dPGJ2 would have on peripheral leukocytes.

• Aims: NF-κB – To determine if NF-κB is activated in PBMCs at term and in labour– To determine if 15dPGJ2 can inhibit NF-κB in PBMCs

Th1/Th2 interleukins– To determine if the pro inflammatory cytokines IFN-γ and TNF-α are increased

at term and in labour– To determine if 15dPGJ2 can inhibit the Th1 cytokines IFN-γ and TNF-α– To determine if 15dPGJ2 can enhance the Th2 cytokine IL-4 via CRTH2

Hypothesis and aims

Th1/Th2 interleukins– To determine if the pro inflammatory cytokines IFN-γ and TNF-α are increased

at term and in labour– To determine if 15dPGJ2 can inhibit the Th1 cytokines IFN-γ and TNF-α– To determine if 15dPGJ2 can enhance the Th2 cytokine IL-4 via CRTH2

Hypothesis and aims

Th1/Th2 interleukins– To determine if the pro inflammatory cytokines IFN-γ and TNF-α are

increased at term and in labour– To determine if 15dPGJ2 can inhibit the Th1 cytokines IFN-γ and TNF-α– To determine if 15dPGJ2 can enhance the Th2 cytokine IL-4 via CRTH2

Experimental design

• Blood was collected in accordance with Ethical approval from South East London Ethical Committee Ref 10/H0805/54 (n>4 for each group)– Non pregnant controls– 28 weeks gestation– Term pre labour– Term in labour

NF-κB studies:• Whole cell lysate was used to analyse Phosphorylated p65 by Western analysis in non stimulated or

PMA (50ng/ml)/Ionomycin (0.5µg/ml) stimulated PBMCs, with or without a 2 hour incubation step of vehicle or 15dPGJ2 (32 µm)

Interleukin studies: • PBMCs were cultured overnight and stimulated with PMA/Ionomycin for 4 hours, with a pre-incubation

step of 2 hrs with vehicle or 15dPGJ2. • Surface cell staining: CRTH2 and CD4 • Intracellular staining: IFN-γ, TNF-α and IL-4• Detection by flow cytometry

Figure 9. Peripheral blood mononuclear cells were isolated using Ficoll-Paque TM

Figure 10. Detection was by flow cytometry, with gating on lymphocytes

NF-κB activity during pregnancy and in labour in peripheral blood mononuclear cells

Figure 11. NF-κB p65 phosphorylation in peripheral blood mononuclear cells at different gestational time points

PBMCs were isolated with Ficoll Paque and whole cell lysate was prepared from the lymphocyte halo. 15ug of protein was run on a western. Densitometry analysis of Phospho p65 was performed against B actin loading control

• There is no difference in NF-κB activity in peripheral mononuclear cells of non pregnancy women and women at 28 weeks of gestation

• There is no increase in NF-κB activity in women in term labour compared to term not in labour.

NF-κB activity during pregnancy and in labour in peripheral blood mononuclear cells

Figure 11. NF-κB p65 phosphorylation in peripheral blood mononuclear cells at different gestational time points

PBMCs were isolated with Ficoll Paque and whole cell lysate was prepared from the lymphocyte halo. 15ug of protein was run on a western. Densitometry analysis of Phospho p65 was performed against B actin loading control

• There is no difference in NF-κB activity in peripheral mononuclear cells of non pregnancy women and women at 28 weeks of gestation

• There is no increase in NF-κB activity in women in term labour compared to term not in labour.

15dPGJ2 reduces NF-κB in peripheral blood mononuclear cells

Figure 12. The effect of 15dPGJ2 on NF-κB (phospho-p65 ) in PBMCs

A Cultured PBMCS were pre-incubated with 32um of 15dPGJ2 or vehicle control

B Results are summarized in the graph, n=4.

C Treated cells were stimulated with 50ng/ml of PMA and 0.5ug/ml of Ionomycin. 15ug of whole cell lysate was run on a western

15dPGJ2 inhibits basal phospho-p65

Independent of CRTH2

NS V J2 J2/x481 x481

β actin

The effect of 15dPGJ2 on Phospho-p65

**Non stim

PMA/Iono

PMA/Iono 15dPGJ2

15dPGJ2 reduces PMA/Ionomycin stimulated phospho-p65 at all gestational time points

Independent of CRTH2

NS V J2 J2/x481 x481

β actin

The effect of 15dPGJ2 on Phospho-p65

**Non stim

PMA/Iono

PMA/Iono 15dPGJ2

Independent of CRTH2 The effect of 15dPGJ2 on Phospho-p65

**Non stim

PMA/Iono

PMA/Iono 15dPGJ2

NS V J2 J2/x481 x481

β actin

IFN-γ and TNF-α production is reduced in pregnancy in T helper cells

Figure 13. The percentage of T helper cells producing the Th1 interleukins IFN-γ and TNF-α during pregnancy and in labourCultured PBMCS were stimulated with 50ng/ml of PMA and 0.5ug/ml of Ionomycin. Cells were stained with CD4 and either IFN -γ or TNF-α antibodies analysed by flow cytometry n=4.

• The percentage of Th1 cells producing IFN-γ and TNF-α (non stimulated and PMA stimulated) in pregnancy was reduced • There was no increase seen in labouring women

IFN-γ and TNF-α production is reduced in pregnancy in T helper cells

Figure 13. The percentage of T helper cells producing the Th1 interleukins IFN-γ and TNF-α during pregnancy and in labourCultured PBMCS were stimulated with 50ng/ml of PMA and 0.5ug/ml of Ionomycin. Cells were stained with CD4 and either IFN -γ or TNF-α antibodies analysed by flow cytometry n=4.

• The percentage of Th1 cells producing IFN-γ and TNF-α (non stimulated and PMA stimulated) in pregnancy was reduced • There was no increase seen in labouring women

15dPGJ2 suppresses the Th1 cytokines IFN-γ and TNF-α

Figure 14. The effect of 15dPGJ2 on PMA/Ionomycin stimulated IFN-γA) A representative dot plot of CD4+/IFN-γ+ cells. An increase in IFN gamma is seen with PMA stimulation which reduces significantly with 15dPGJ2 treatmentB) Histogram showing the effect of 15dPGJ2 on IFN gamma in CD4 positive cells (T helper cells)

NS PMA PMA/15dPGJ2

IFN-γ- FITC

CD4-APC

Figure 15. The effect of 15dPGJ2 on IFN-γ and TNF-α production in T helper cellsA reduction in mean fluorescence intensity of IFN-γ was seen at all gestations (p<0.05 in non pregnant, 28 weeks and at term). A reduction was also seen in TNF-α production, but this did not reach statistical significance (n=4).

NS PMA PMA/15dPGJ2

IFN-γ- FITC

CD4-APC

NS PMA PMA/15dPGJ2

IFN-γ- FITC

CD4-APC

NS PMA PMA/15dPGJ2

IFN-γ- FITC

CD4-APC

15dPGJ2 does not increase the Th2 cytokine IL-4

Figure 17. The effect of 15dPGJ2 on IL-4 production in CRTH2 + lymphocytesNo increase in IL-4 production was seen with the CRTH2 agonist 15dPGJ2 (n=4).

Figure 16. Representative dot plot of PMA stimulated IL-4 in CRTH2+ cells

Poster T-238 Sykes et al

• PMA stimulates IL-4 in CRTH2 positive cells

• No increase in IL-4 production with 15dPGJ2

Conclusion

• The anti inflammatory prostaglandin 15dPGJ2 is capable of modulating the peripheral Th1 response, possibly via NF-κB

• 15dPGJ2 does not modulate the Th2 cytokine profile of IL-4 in CRTH2 positive lymphocytes (Th2 cells)

15dPGJ2

Conclusion

• The anti inflammatory prostaglandin 15dPGJ2 is capable of modulating the peripheral Th1 response, possibly via NF-κB

• 15dPGJ2 does not modulate the Th2 cytokine profile of IL-4 in CRTH2 positive lymphocytes (Th2 cells)

15dPGJ2

Conclusion

• 15dPGJ2, consistent with the local effects seen on myocytes and amniocytes, also has peripheral anti-inflammatory effects by reducing NF-κB and modulating the Th1:Th2 ratio

• It therefore remains a potential anti- inflammatory agent for the prevention of both preterm labour and inflammation induced adverse neonatal outcomes

15dPGJ2

Conclusion

• 15dPGJ2, consistent with the local effects seen on myocytes and amniocytes, also has peripheral anti-inflammatory effects by reducing NF-κB and modulating the Th1:Th2 ratio

• It therefore remains a potential anti- inflammatory agent for the prevention of both preterm labour and inflammation induced adverse neonatal outcomes

15dPGJ2

Thanks to.........

The co-authors; David MacIntyre, Research AssociateSathana Ponnampalam, Clinical ResearcherXiao J Yap, MSc project

The support from our research group; Dr Yooni Lee, Sung Hye Kim, Dr Manju Chandiramani, Dr Jenny Loudon, Dr Vasso Terzidou

My supervisors; Mr TG Teoh and Professor Phillip Bennett

To Wellbeing of Women for funding this research

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